U.S. patent application number 12/681378 was filed with the patent office on 2010-08-26 for novel method for preparing nanoparticles covered with a gem-bisphosphonate stabilizing layer coupled to hydrophilic biodistribution ligands.
This patent application is currently assigned to GUERBET. Invention is credited to Marc Port, Olivier Rousseaux.
Application Number | 20100215586 12/681378 |
Document ID | / |
Family ID | 39242259 |
Filed Date | 2010-08-26 |
United States Patent
Application |
20100215586 |
Kind Code |
A1 |
Port; Marc ; et al. |
August 26, 2010 |
NOVEL METHOD FOR PREPARING NANOPARTICLES COVERED WITH A
GEM-BISPHOSPHONATE STABILIZING LAYER COUPLED TO HYDROPHILIC
BIODISTRIBUTION LIGANDS
Abstract
A novel method for preparing nanoparticles for medical imaging,
including a metal core, an organic stabilizing layer containing
gem-bisphosphonate compounds and at least one hydrophilic
biodistribution ligand.
Inventors: |
Port; Marc; (Deuil La Barre,
FR) ; Rousseaux; Olivier; (Senlis, FR) |
Correspondence
Address: |
YOUNG & THOMPSON
209 Madison Street, Suite 500
Alexandria
VA
22314
US
|
Assignee: |
GUERBET
Villepinte
FR
|
Family ID: |
39242259 |
Appl. No.: |
12/681378 |
Filed: |
October 6, 2008 |
PCT Filed: |
October 6, 2008 |
PCT NO: |
PCT/FR08/51802 |
371 Date: |
April 14, 2010 |
Current U.S.
Class: |
424/9.32 ;
424/9.3 |
Current CPC
Class: |
C09B 11/24 20130101;
C09B 23/0066 20130101; C09B 23/0033 20130101; A61K 49/186 20130101;
A61K 49/0428 20130101; C09B 23/083 20130101; B82Y 5/00 20130101;
A61K 49/1842 20130101 |
Class at
Publication: |
424/9.32 ;
424/9.3 |
International
Class: |
A61K 49/18 20060101
A61K049/18 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 5, 2007 |
FR |
0758103 |
Claims
1-12. (canceled)
13. A process for preparing metallic nanoparticles comprising a
metallic core N covered with an organic stabilizing layer coupled
to at least one hydrophilic ligand having an effect on the
stability/biodistribution of the nanoparticles, comprising the
steps of: a) preparing the metallic core N of the metallic
nanoparticles; b) preparing targeting elements of formula S--C in
which: S is a gem-bisphosphonate attachment group of formula
X-L-CH(PO.sub.3H.sub.2).sub.2; C is a hydrophilic biodistribution
ligand chosen from amino alcohols or PEGs; c) grafting of the
targeting elements S--C to the core N; where: L represents an
organic group that connects the X functional group to the
gem-bisphosphonate --CH(PO.sub.3H.sub.2).sub.2 functional group;
and X represents a chemical functional group capable of being
coupled to the hydrophilic biodistribution ligand C.
14. The process as claimed in claim 13, in which the metallic core
is chosen from the following: iron hydroxide, hydrated iron oxide,
ferrite, or mixed iron oxide.
15. The process as claimed in claim 13, in which the hydrophilic
biodistribution ligand is an amino alcohol ligand.
16. The process as claimed in claim 15, in which the amino alcohol
ligand is a compound of formula (II): ##STR00034## in which:
R.sub.1 and R.sub.2 are identical or different and represent an
aliphatic hydrocarbon-based chain comprising from 2 to 6 carbon
atoms, preferably substituted with 6 to 10 hydroxyl groups, or else
with to 8 hydroxyl groups in the case where R.sub.1 and/or R.sub.2
is interrupted by one or more oxygen atoms.
17. The process as claimed in claim 15, in which the amino alcohol
ligand is a compound of formula (IV): ##STR00035## in which: Z is a
bond, CH.sub.2, CH.sub.2CONH or (CH.sub.2).sub.2NHCO; Z' is a bond,
O, S, NQ, CH.sub.2, CO, CONQ, NQCO, NQ-CONQ or CONQCH.sub.2CONQ,
Z'' is a bond, CONQ, NQCO or CONQCH.sub.2CONQ; p and q are
integers, the sum of which is equal to 0 to 3; R.sub.1, R.sub.2,
R.sub.3, R.sub.4 or R.sub.5 each independently represent H, Br, Cl,
I, CONQ.sub.1Q.sub.2 or NQ.sub.1COQ.sub.2; with Q.sub.1 and
Q.sub.2, which are identical or different, chosen from H, a
(C.sub.1-C.sub.8)alkyl group that is monohydroxylated or
polyhydroxylated and/or optionally interrupted by one or more
oxygen atoms, so that Q.sub.1 and Q.sub.2 comprise, between them,
from 4 to 10 OH groups; it being understood that at least one and
at most two of the groups R.sub.1 to R.sub.5 represents
CONQ.sub.1Q.sub.2 or NQ.sub.1COQ.sub.2; or else R.sub.1, R.sub.3,
R.sub.5 each independently represent H, Br, Cl or I and R.sub.2 and
R.sub.4 represent: ##STR00036## in which: R'.sub.1, R'.sub.3 and
R'.sub.5, which are identical or different, represent H, Br, Cl or
I; Q.sub.1 and Q.sub.2 have the same meaning as above; Z''' is a
group chosen from CONQ, CONQCH.sub.2CONQ, CONQCH.sub.2, NQCONQ,
CONQ(CH.sub.2).sub.2NQCO; and Q is H or (C.sub.1-C.sub.4)alkyl,
said alkyl groups possibly being linear or branched and optionally
being hydroxylated.
18. The process as claimed in claim 13, in which the hydrophilic
biodistribution ligand is a polyethylene glycol ligand.
19. The process as claimed in claim 18, in which the polyethylene
glycol ligand is an amino polyethylene glycol of formula (III):
##STR00037## in which: R.sub.1 and R.sub.2, which are identical or
different, represent H, an alkyl group or a polyethylene glycol
chain of formula
--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.k--CH.sub.2OR.sub.3, it
being understood that at least one of the groups R.sub.1, R.sub.2
represents a polyethylene glycol chain; in which k varies from 2 to
100; and R.sub.3 is chosen from H, C.sub.1-C.sub.6 alkyl or
--(CO)Alk, with "alk" denoting a C.sub.1-C.sub.6 alkyl group.
20. The process as claimed in claim 13, in which one portion of the
hydrophilic biodistribution ligands C are amino alcohol ligands and
another portion are polyethylene glycol ligands.
21. The process as claimed in claim 13, in which grafted to the
core are, on the one hand, targeting elements S--C and, on the
other hand, stabilizing groups S that do not bear biodistribution
ligands.
22. The process as claimed in claim 13, in which the degree of
grafting of the targeting elements to the core is from 1 to 10%,
advantageously 1, 2, 3, 5 or 10%.
23. The process as claimed in claim 13, comprising, in addition,
the grafting of elements S--C-T, where C is a polyethylene glycol
ligand and T represents a chromophore group.
24. A targeting element of formula S--C in which: S is a
gem-bisphosphonate linking group of formula
X-L-CH(PO.sub.3H.sub.2).sub.2; where: L represents an organic group
that connects the X functional group to the gem-bisphosphonate
--CH(PO.sub.3H.sub.2).sub.2 functional group; X represents a
chemical functional group capable of being coupled to the
hydrophilic ligand C; and C is a hydrophilic biodistribution ligand
chosen from amino alcohols or PEGs.
Description
[0001] The invention relates to a novel process for preparing
nanoparticles for medical imaging comprising a metallic core, an
organic stabilizing layer and at least one ligand for targeting a
pathological tissue.
[0002] Metallic nanoparticles used in diagnostic imaging,
especially in magnetic resonance imaging MRI are known, that
comprise a metallic core, covered with a stabilizing organic layer
coupled, where appropriate, to biological targeting ligands.
[0003] Among these nanoparticles, metallic nanoparticles commonly
denoted by USPIO are especially known, which are very small
particles of iron oxide, including, in particular, magnetite
(Fe.sub.3O.sub.4), maghemite (.gamma.-Fe.sub.2O.sub.3) and other
magnetic mineral compounds of transition elements, having a size of
less than approximately 100-150 nm.
[0004] In order to obtain colloidal solutions of magnetic particles
that are stable in a physiological medium, it is necessary to
condition the surface of the magnetic particles. In order to do
this, the particle is covered with a stabilizing organic layer
constituted of macromolecules such as carbohydrates, for instance
dextran, or of small organic molecules such as carboxylic
acids.
[0005] In order to obtain relevant information for medical imaging
diagnosis, it is highly advantageous to couple the latter to
suitable targeting ligands, so that the particles bind to and/or
are recognized by target tissues or cells. This recognition may
advantageously be provided using ligands that have an effect on the
biodistribution of the product, for example via a phagocytosis-type
mechanism of the particle by cells of the immune system such as
macrophages. These ligands are, for example, hydrophilic groups
such as amino alcohol groups, or compounds of polyethylene glycol
(PEG) type.
[0006] Document WO 2004/058275 describes the synthesis of compounds
using, as a stabilizing/attachment layer, a layer of
gem-bisphosphonate type, and, as a ligand, among the many possible
ligands, hydrophilic groups that have an effect on the
biodistribution (biodistribution ligands). Compounds are in
particular described that are in the form of a metallic core N
covered with targeting elements of formula S--C, in which: [0007] S
is a gem-bisphosphonate group grafted to the core and of formula
(I):
[0007] X-L-CH(PO.sub.3H.sub.2).sub.2 (I) [0008] C is a hydrophilic
ligand (coupled to the X functional group) of amino alcohol type
and/or of PEG type; [0009] where: [0010] L represents an organic
group that connects the X functional group to the
gem-bisphosphonate --CH(PO.sub.3H.sub.2).sub.2 functional group;
and [0011] X represents a chemical functional group capable of
being coupled to the hydrophilic ligand C.
[0012] For the preparation of these compounds, the prior process
(used generally for USPIOs) schematically comprises the following
steps: [0013] preparation of the metallic core N of the metallic
nanoparticles; [0014] coating of the core N with the
gem-bisphosphonate stabilizing layer of formula:
[0014] X-L-CH(PO.sub.3H.sub.2).sub.2 (I) [0015] coupling of the
particle obtained with the hydrophilic group or groups.
[0016] It is sought to improve this process in order to obtain
compounds for which the amount of ligand grafted is perfectly
mastered, in order to optimize and to control, with
reproducibility, the degree of grafting onto the core, to avoid
purification steps and to simplify the pharmaceutical control by
minimizing the risks of bacterial or pyrogen contamination, and
thus to obtain an efficient production of the product on an
industrial scale.
[0017] Furthermore, in particular in the case of biodistribution
ligands of amino alcohol or polyethylene glycol type, which are
described in detail below, an additional problem lies in the
required amount of ligand to be used in the process described in WO
2004/058275. Indeed, in order to obtain, in the end, a degree of
coverage of targeting elements S--C that is advantageously high,
especially a degree of coverage of more than 80% of the possible
sites of attachment to the core, it was necessary, in this prior
process, to use a large amount of ligand C in excess (it was
necessary to add, to one equivalent of compound N--S
[core+gem-bisphosphonate covering], around 5 equivalents of
hydrophilic ligand C), hence high industrial costs. It was
particularly useful to solve this problem for complex and expensive
amino alcohols such as those of formula (IV), especially the amino
alcohols AAG1 and derivatives thereof described below in the
present application.
[0018] The applicant has succeeded in solving these technical
problems by virtue of a preparation process (denoted by reverse
pathway) according to which elements are prepared that are
constituted by one or more hydrophilic ligands chemically coupled
with organic gem-bisphosphonate linkage groups, then these elements
[linkage group-ligand] are coupled to the metallic nanoparticles.
The organic linkage groups will belong to or form the stabilizing
(or attachment) layer.
[0019] For this purpose, the invention relates to a process for
preparing metallic nanoparticles, for medical imaging in
particular, comprising a metallic core N covered with an organic
stabilizing layer coupled to at least one hydrophilic ligand having
an effect on the stability/biodistribution of the nanoparticles,
the process comprising the steps of: [0020] a) preparing the
metallic core N of the metallic nanoparticles; [0021] b) preparing
targeting/stabilizing elements of formula S--C in which: [0022] S
is a gem-bisphosphonate attachment group of formula
X-L-CH(PO.sub.3H.sub.2).sub.2; [0023] C is a hydrophilic
biodistribution ligand advantageously chosen from amino alcohols or
PEGs; [0024] c) grafting of at least one targeting element to the
core N.
[0025] For the sake of simplicity, in the application the
expression "targeting elements" will be used for the expression
targeting/stabilizing elements.
[0026] The expression "grafting of at least one targeting element
to the core N" is understood to mean that targeting assemblies of
the same structure, or several targeting assemblies for which the S
groups and/or the C groups do not have the same formula, are
grafted.
[0027] The advantage of having different S groups is, in
particular, to be able to increase or reduce the hydrodynamic size
of the contrast agent (by varying the size of the L groups), which
makes it possible to help to optimize the biodistribution of the
product as a function of the diagnostic indication in question.
[0028] According to some embodiments, the targeting elements are
amino alcohols.
[0029] According to some embodiments, the targeting elements are
PEGs.
[0030] According to some embodiments, one portion of the targeting
elements are amino alcohols and another portion of the targeting
elements are PEGs. The ligands may be identical or different
between the targeting elements grafted.
[0031] According to some embodiments, the metallic nanoparticles
obtained after grafting will thus have, for example, 10 to 90% of
targeting elements with a hydrophilic ligand that has an
advantageous effect on the biodistribution and/or the stability of
the particle (an amino alcohol, a PEG branch, several different
amino alcohols) and the balance (90 to 10%) of the targeting
elements with a different ligand.
[0032] According to some embodiments, grafted to the core are, on
the one hand, targeting elements S--C (bearing hydrophilic groups)
and, on the other hand, stabilizing groups S that do not bear
biodistribution ligands. For example, there will be 5 to 95% of
S--C groups and the balance (95 to 5%) of S groups.
[0033] The table at the end of the detailed description illustrates
various possibilities.
[0034] These amounts correspond to the degree of coverage of the
core by the elements S or S--C (at the possible attachment sites,
typically the protonated sites located at the surface of the
nanoparticle). The percentages are thus expressed as the number of
S--C or S molecules per number of attachment sites available on the
core. Thus, for a degree of coverage of 100%, the surface of the
core will be substantially completely covered with S--C and/or S
elements (for example 80% of S--C groups and 20% of S groups). This
degree of coverage is thus different from the degree of grafting
described below.
[0035] The metallic nanoparticles have a hydrodynamic diameter of
the order of 10 to 100 nm, and especially of 10 to 50 nm.
[0036] Each S group of a targeting assembly comprises at least one
core linkage portion and at least one chemical functional group X
for coupling with a ligand C, more specifically for covalent
bonding to a reactive functional group of the ligand.
[0037] Steps a) and b) may be carried out in any order but before
step c).
[0038] The assembly of the S groups grafted to the core N
constitutes the attachment (stabilizing) layer. The degree of
grafting (molar percentage of compound S--C and/or S per mole of
iron; the degree of grafting is determined from phosphorus assays)
of the targeting elements S--C and/or S to the core N is typically
between 0.5 and 10%, especially 1 to 5%, for example 1, 2, 3, 5 or
10%, for a crystal size of the core of the order of 7-8 nm.
[0039] According to some embodiments, in addition to the targeting
elements S--C, groups are also grafted that have an effect on the
stability of the nanoparticle, for example hydroxymonocarboxylic
acids, for example chosen from the following: gluconic acid, oxalic
acid, mandelic acid, 4-hydroxy-3-methoxymandelic acid, lactobionic
acid, alpha-hydroxyhippuric acid, methyl-2-hydroxybutyric acid,
glycolic acid, N-acetylneuraminic acid, or phosphoenolpyruvic
acid.
[0040] Thus, very advantageously, it is possible to perfectly
control the degree of grafting of the nanoparticles with compounds
that bear ligands, which is very useful for the cost, the analysis
and the characterization and the control of the physiological
effectiveness of the product. The manufacture of the targeting
elements S--C is furthermore completely controlled, in particular
their purity before grafting, which is important for industrial
manufacture.
[0041] The core N will now be described more precisely. The
metallic core of the nanoparticles prepared is typically composed,
completely or partly, of iron hydroxide; hydrated iron oxide; mixed
iron oxides such as mixed oxides of iron with cobalt, nickel,
manganese, beryllium, magnesium, calcium, barium, strontium,
copper, zinc or platinum; or of a mixture of the latter. The term
"ferrite" denotes the iron oxides of general formula
[xFe.sub.2O.sub.3.yMO.sub.z], where M denotes a metal that can be
magnetized under the effect of a magnetic field such as Fe, Co, Ru,
Mg or Mn, it being possible for the magnetizable metal to
optionally be radioactive. Preferably, the magnetic particles of
the compositions of the invention comprise a ferrite, especially
maghemite (.gamma.-Fe.sub.2O.sub.3) and magnetite
(Fe.sub.3O.sub.4), or else ferrites mixed with cobalt
(Fe.sub.2CoO.sub.4) or with manganese (Fe.sub.2MnO.sub.4). The core
of the nanoparticle was rendered acid to facilitate the coupling of
the S--C elements. The process for preparing the acid core (with a
step using nitric acid) is described in detail in document WO
2004/058275 (US 2004/253181, especially paragraphs 331 to 339, page
19). It is recalled that this process with an acidification step
(at a highly acidic pH--typically between 1 and 3) before the
attachment of the S and/or S--C groups, makes it possible to obtain
particularly advantageous particles, the polydispersity of which is
completely controlled and which are in a stable colloidal
solution.
[0042] The L groups will now be described. Preferably, the L
linkage group is a divalent group, preferably chosen from: [0043]
an aliphatic; alicyclic; alicyclic-aliphatic; aromatic; or
aromatic-aliphatic group, said aliphatic, alicyclic and aromatic
groups possibly being optionally substituted with a methyl,
hydroxy, methoxy, acetoxy or amido group or a chlorine, iodine or
bromine atom; [0044] an -L.sub.1-NHCO-L.sub.2 group where L.sub.1
and L.sub.2 are either identical or different and represent an
aliphatic; alicyclic; aromatic; alicyclic-aliphatic or
aromatic-aliphatic group, said groups possibly being optionally
substituted with a methyl, hydroxy, methoxy, acetoxy or amido group
or a chlorine, iodine or bromine atom.
[0045] An aliphatic group denotes here a linear or branched
hydrocarbon-based chain preferably comprising from 1 to 16 carbon
atoms, better still from 1 to 6 carbon atoms. Preferably, the
aliphatic group denotes an alkyl group. Examples thereof are
especially methyl, ethyl, propyl, isopropyl, butyl, tert-butyl,
isobutyl, pentyl and hexyl radicals.
[0046] The term "alicyclic" denotes a cyclic hydrocarbon-based
chain preferably comprising from 3 to 8 carbon atoms, preferably a
cycloalkyl group. By way of example, mention will especially be
made of cyclopropyl and cyclohexyl.
[0047] The term "aromatic" represents an aromatic monocyclic or
polycyclic hydrocarbon-based group preferably comprising from 5 to
20, better still from 6 to 18, carbon atoms. Examples thereof are
especially phenyl and 1-naphthyl or 2-naphthyl radicals. According
to one particular variant, an "aromatic" group within the meaning
of the invention may incorporate one or more heteroatoms such as
sulfur, oxygen or nitrogen. In this particular case, the "aromatic"
group denotes a monocyclic or polycyclic heteroaromatic group.
[0048] The "aliphatic-alicyclic" and "aliphatic-aromatic" groups
represent aliphatic chains corresponding to the aforementioned
definition, substituted respectively with an alicyclic or aromatic
group as defined above. By way of example of an aliphatic-aromatic
group, mention may especially be made of benzyl.
[0049] According to one preferred variant, L represents an
optionally substituted phenylene group, the X and
gem-bisphosphonate groups possibly being in the ortho, meta or para
position.
[0050] According to one particularly preferred embodiment, L
represents a substituted or unsubstituted aliphatic group and more
preferably a --(CH.sub.2).sub.p-- group, where p is an integer from
1 to 5.
[0051] According to another preferred embodiment, L represents an
L.sub.1-CONH-L.sub.2 group and more preferably a
--(CH.sub.2).sub.n--NHCO--(CH.sub.2).sub.m-- group where n and m
represent an integer from 0 to 5.
[0052] The X end of the gem-bisphosphonate compound of formula (I)
is chosen so that it is capable of reacting and of forming a
covalent bond with a functional group present on the biovector. For
more information concerning these couplings, reference may
especially be made to the work Bioconjugate techniques, Greg T.
Hermanson, 1995, Publisher: Academic, San Diego, Calif.
[0053] As preferred X groups, mention may especially be made of:
[0054] --COOH, [0055] --NH.sub.2, --NCS, --NH--NH.sub.2, --CHO,
alkylpyrocarbonyl (--CO--O--CO-alk), acylazidyl (--CO--N.sub.3),
iminocarbonate (--O--C(NH)--NH.sub.2), vinylsulfuryl
(--S--CH.dbd.CH.sub.2), pyridyldisulfuryl (--S--S-Py), haloacetyl,
maleimidyl, dichlorotriazinyl, halogen, [0056] the group of
formula:
[0056] ##STR00001## [0057] the --COOH and --NH.sub.2 groups being
particularly preferred.
[0058] The term "alk" denotes, within the meaning of the present
description, a C.sub.1-C.sub.6 alkyl radical, the term "Py"
denoting, for its part, a pyridyl radical.
[0059] The maleimidyl radical denotes a cyclic radical of
formula:
##STR00002##
[0060] The dichlorotriazinyl radical denotes a radical of
formula:
##STR00003##
[0061] Among the halogen groups, mention may especially be made of
chlorine, bromine, fluorine and iodine, chlorine and bromine being
particularly preferred.
[0062] The term "haloacetyl", within the meaning of the present
description, is understood to mean an acetyl radical
CH.sub.3--CO--, one of the hydrogen atoms of which is substituted
with a halogen atom, said halogen atom being as defined above.
[0063] Preferably, X represents a --COOH or --NH.sub.2 group and L
a substituted or unsubstituted aliphatic group, better still a
--(CH.sub.2).sub.p-- group, where p is an integer from 1 to 5.
[0064] The compound of formula (Ia):
HOOC--(CH.sub.2).sub.2--CH(PO.sub.3H.sub.2).sub.2 (Ia)
is most particularly preferred.
[0065] According to another preferred embodiment, L represents an
L.sub.1-CONH-L.sub.2 group and more preferably a
--(CH.sub.2).sub.n--NHCO--(CH.sub.2).sub.m-- group where n and m
represent an integer from 0 to 5 and X represents --COOH or
--NH.sub.2.
[0066] Of course, also falling within the context of the invention
is the coupling of the X functional group and of the biovector in
an indirect manner, that is to say via a homobifunctional or
heterobifunctional reagent. By way of illustration of the
homobifunctional reagent, glutaraldehyde may be suitable for
carrying out the coupling, for example, of an X.dbd.NH.sub.2
functional group with an --NH.sub.2 functional group of the
biovector.
[0067] According to one preferred variant of the invention, the X
functional groups form a covalent bond L.sub.3 with the biovector,
of the type:
[0068] --CONH--, --COO--, --NHCO--, --OCO--, --NH--CS--NH--,
--C--S--, --N--NH--CO--, --CO--NH--N--, --CH.sub.2--NH--,
--N--CH.sub.2--, --N--CS--N--, --CO--CH.sub.2--S--,
--N--CO--CH.sub.2--S--, --N--CO--CH.sub.2--CH.sub.2--S--,
--CH.dbd.NH--NH--, --NH--NH.dbd.CH--, --CH.dbd.N--O--,
--O--N.dbd.CH-- or corresponding to the following formulae:
##STR00004##
[0069] All or some, and typically of the order of 50 to 100%,
especially 50, 60, 70, 80, 80 or 95% of the X functional groups of
the gem-bisphosphonate compound are coupled to a biodistribution
ligand.
[0070] Preferably, the hydrophilic biodistribution ligand is an
amino alcohol or polyethylene glycol (also known as PEG)
ligand.
[0071] The term "amino alcohol" according to the present
application is understood to mean a ligand comprising an amine
functional group bearing at least one aliphatic hydrocarbon-based
chain comprising from 2 to 10 carbon atoms, said hydrocarbon-based
chain being substituted by several hydroxyl groups, especially by 4
to 10 hydroxyl groups.
[0072] According to one preferred embodiment, the amino alcohol
ligands are compounds of general formula (II):
##STR00005##
[0073] in which:
[0074] R.sub.1 and R.sub.2 are identical or different and represent
an aliphatic hydrocarbon-based chain comprising from 2 to carbon
atoms, preferably substituted with 6 to 10 hydroxyl groups, or else
with 4 to 8 hydroxyl groups in the case where R.sub.1 and/or
R.sub.2 is interrupted by one or more oxygen atoms.
[0075] As an example of an amino alcohol ligand of formula (II),
mention may especially be made of the ligands for which R.sub.1 and
R.sub.2 each independently represent a
--(CH.sub.2)--(CHOH).sub.4--CH.sub.2OH or
--(CH.sub.2)--CHOH--CH.sub.2OH group, in particular those for which
R.sub.1 represents a --(CH.sub.2)--(CHOH).sub.4--CH.sub.2OH or
--(CH.sub.2)--CHOH--CH.sub.2OH group and R.sub.2 a
--CH.sub.2--(CHOH).sub.4--CH.sub.2OH group.
[0076] According to another preferred embodiment, the amino alcohol
ligands are compounds of formula (IV):
##STR00006## [0077] in which: [0078] Z is a bond, CH.sub.2,
CH.sub.2CONH or (CH.sub.2).sub.2NHCO; [0079] Z' is a bond, O, S,
NQ, CH.sub.2, CO, CONQ, NQCO, NQ-CONQ or CONQCH.sub.2CONQ, [0080]
Z'' is a bond, CONQ, NQCO or CONQCH.sub.2CONQ; [0081] p and q are
integers, the sum of which is equal to 0 to 3 (with, according to
one advantageous variant, p=q=0); [0082] R.sub.1, R.sub.2, R.sub.3,
R.sub.4 or R.sub.5 each independently represent H, [0083] Br, Cl,
I, CONQ.sub.1Q.sub.2 or NQ.sub.1COQ.sub.2; [0084] with Q.sub.1 and
Q.sub.2, which are identical or different, chosen from H, a
(C.sub.1-C.sub.8)alkyl group that is monohydroxylated or
polyhydroxylated and/or optionally interrupted by one or more
oxygen atoms, so that Q.sub.1 and Q.sub.2 comprise, between them,
from 4 to 10 OH groups; [0085] it being understood that at least
one and at most two of the groups R.sub.1 to R.sub.5 represents
CONQ.sub.1Q.sub.2 or NQ.sub.1COQ.sub.2; [0086] or else R.sub.1,
R.sub.3, R.sub.5 each independently represent H, Br, Cl or I and
R.sub.2 and R.sub.4 represent:
[0086] ##STR00007## [0087] in which: [0088] R'.sub.1, R'.sub.3 and
R'.sub.5, which are identical or different, represent H, Br, Cl or
I; [0089] Q.sub.1 and Q.sub.2 have the same meaning as above;
[0090] Z''' is a group chosen from CONQ, CONQCH.sub.2CONQ,
CONQCH.sub.2, NQCONQ, CONQ(CH.sub.2).sub.2NQCO; and [0091] Q is H
or (C.sub.1-C.sub.4)alkyl, said alkyl groups possibly being linear
or branched and optionally being hydroxylated.
[0092] Preferably, Z is CH.sub.2.
[0093] Preferably, p=q=0.
[0094] Preferably, Z'' is CONH.
[0095] Preferably, R.sub.2 and R.sub.4 represent
CONQ.sub.1Q.sub.2.
[0096] Preferably, R.sub.1, R.sub.3, R.sub.5 represent Br.
[0097] Preferably, Q.sub.1 and Q.sub.2 each independently represent
a --(CH.sub.2)-- (CHOH).sub.4--CH.sub.2OH or
--(CH.sub.2)--CHOH--CH.sub.2OH group, in particular a
--(CH.sub.2)--(CHOH).sub.4--CH.sub.2OH group.
[0098] According to one particularly preferred embodiment, the
amino alcohol ligand of formula (IV) is the compound:
##STR00008##
[0099] Preferably, the amino alcohol ligands according to the
invention are coupled via their --NH-- or --NH.sub.2 amine
functional group to the X functional group of the attachment groups
S of formula X-L-CH(PO.sub.3H.sub.2).sub.2, so that the hydroxyl
functional groups remain free, thus preserving their hydrophilic
nature.
[0100] The expression "polyethylene glycol", within the meaning of
the present application, generally denotes compounds comprising a
--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.k--CH.sub.2OR.sub.3 chain
in which k varies from 2 to 100 (for example: 2, 4, 6, 10 or 50)
and R.sub.3 is chosen from H, alkyl or --(CO)Alk, the term "alkyl"
or "alk" denoting a linear or branched hydrocarbon-based aliphatic
group having around 1 to 6 carbon atoms in the chain.
[0101] The expression "polyethylene glycol" as used here,
encompasses, in particular, the amino polyethylene glycol compounds
of formula (III):
##STR00009## [0102] in which: [0103] R.sub.1 and R.sub.2, which are
identical or different, represent H, an alkyl group or a
polyethylene glycol chain of formula
--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.k--CH.sub.2OR.sub.3, it
being understood that at least one of the groups R.sub.1, R.sub.2
represents a polyethylene glycol chain; in which k varies from 2 to
100 (for example: 2, 4, 6, 10 or 50); and [0104] R.sub.3 is chosen
from H, alkyl or --(CO)Alk, the term "alkyl" or "alk" denoting a
linear or branched hydrocarbon-based aliphatic group having around
1 to 6 carbon atoms in the chain.
[0105] Examples of amino polyethylene glycols are, in particular,
the compounds O-(2-aminoethyl)-O'-methyl polyethylene glycol 1100,
O-(2-aminoethyl)-O'-methyl polyethylene glycol 2000,
O-(2-aminoethyl)-O'-methyl polyethylene glycol 750, the compounds
PEG 340, PEG 750, PEG 1500, PEG 2000 for example.
[0106] It is specified that the grafting of the S--C compounds of
the application to the N core is carried out by the
CH(PO.sub.3H.sub.2).sub.2 part of the S compounds.
[0107] The applicant has noted, in particular, for the amino
alcohol and/or PEG ligands that not only is the synthesis
facilitated and has an improved yield, but in addition that the
final product obtained corresponds very satisfactorily to the
regulatory specifications and to the diagnostic use. The coverage
of almost all of the core by these groups gives the product a
stability and a biodistribution that are particularly advantageous
for these hydrophilic ligands, in particular by improving the
stealth (the product is then advantageously taken up less by the
liver) and the macrophage uptake (the targeting of the macrophages
is improved, with an advantage, in particular, for monitoring
atheromatous plaques, ganglia and other areas of inflammation).
Furthermore, the advantage of controlling the amount of ligand is
understood for products having mixed coverage, for example amino
alcohol ligand and PEG ligand, in order to meet the requirements of
reproducibility of the production batches, of quality and of
pharmaceutical safety.
[0108] Unexpectedly, the novel reverse pathway process of the
applicant makes it possible to divide, by a factor of 3 to 10, the
amount of biodistribution ligand, in particular of such amino
alcohols. The following table illustrates this result, by taking
the example of the compound from Example 14 using, as an amino
alcohol, the compound from Example 4.
TABLE-US-00001 Direct pathway (prior art) Reverse pathway Amount of
amino 5 to 10 1.2 to 2.2 alcohol ligand to be equivalents
equivalents added in order to obtain a coverage of S--C elements
greater than 90% Degree of grafting (in 1.2% 2% % of amino alcohol
ligand/mol of Fe) with the amino alcohol from Example 4 Amount of
amino 0.2 mol 0.025 mol alcohol (in mol of amino alcohol) used to
obtain this degree of grafting.
[0109] Thus, the applicant has succeeded in solving the two-fold
problem of controlling the degree of grafting of the ligands and of
reducing the amount of ligand to be used.
[0110] According to one particular embodiment, the process
according to the invention also comprises the grafting of S--C-T
elements, where C is a polyethylene glycol ligand and T represents
a chromophore group.
[0111] The term "chromophore" is understood to mean a colored
group, that is to say one that is capable of absorbing the energy
of photons in one range of the visible spectrum whilst the other
wavelengths are transmitted or scattered.
[0112] As an example of a chromophore that can be used according to
the invention, mention may especially be made of
4-(amino)fluorescein hydrochloride.
[0113] The invention also relates to the use of the nanoparticles
obtained by the process of the applicant for the preparation of a
diagnostic or therapeutic composition. The nanoparticles are, in
particular, used as a contrast agent of nanoparticle composition
type as described in detail in document WO 2004/058275, for MRI
imaging or an X-ray scanner.
[0114] According to some embodiments, the particles are carried in
systems for the release of active principles, such as encapsulation
systems of the solid lipid nanoparticle or liposome type which may
also contain, in addition to the nanoparticles used as a diagnostic
agent, therapeutic active principles.
[0115] Another subject of the invention are the S--C targeting
elements as defined above, that can be used according to the
process of the invention.
[0116] The invention is illustrated using the following detailed
examples.
[0117] In what follows, the abbreviations M, M/L, theoretical M, N
and M/z, ES.sup.+, ES, kD and TLC have the same meanings as in
document WO 2004/058275 (US 2004/253181): [0118] M or M/L: molar
concentration (mol/liter). [0119] Theoretical M: theoretical
molecular mass. [0120] N: normality. [0121] M/z: mass-to-charge
ratio determined by mass spectrometry. [0122] ES.sup.+: positive
mode electrospray. [0123] ES.sup.-: negative mode electrospray.
[0124] TFA: trifluoroacetic acid. [0125] kD: unit of molecular mass
(kiloDalton). [0126] TLC: thin layer chromatography. [0127] Z ave:
hydrodynamic diameter measured by PCS. [0128] Poly .sigma.:
polydispersity measured by PCS.
[0129] The chemical nomenclature which follows is derived from
ACD/NAME software (Advanced Chemistry Development Inc., Toronto,
Canada), according to IUPAC rules.
[0130] Total Iron Assay:
[0131] The iron is assayed by atomic absorption spectroscopy
(VARIAN AA10 spectrophotometer) after mineralization with
concentrated HCl and dilution with respect to a standard range of
ferric ions (0, 5, 10, 15 and 20 ppm).
[0132] Particle Size:
[0133] Hydrodynamic Diameter of the Grafted Particle (Z Ave)=PCS
Size:
[0134] Determined by PCS (Malvern 4700 machine, 488 nm laser at
90.degree.) on a sample diluted to .about.1 millimol with water for
injection, filtered through 0.22 .mu.m.
[0135] PCS=Photon Correlation Spectroscopy Reference:
[0136] R. Pecora in J. of Nano. Res. (2000), 2, p. 123-131.
[0137] Diameter of the Magnetic Particle (p) (Before Grafting):
[0138] Determined by deconvolution of the magnetization curves
(measurements taken on a SQUID magnetometer) at various
temperatures (reference: R. W. Chantrell in IEEE Transactions on
Magnetics (1978), 14(5), p. 975-977).
[0139] Structural Analyses:
[0140] By mass spectroscopy (MICROMASS VG Quattro II machine) with
an electrospray source.
EXAMPLE 1
[0141] A solution of 36 g (0.181 mol) of FeCl.sub.2.4H.sub.2O and
20 ml of 37% HCl in 150 ml of H.sub.2O is introduced into a mixture
consisting of 3 liters of water and 143 ml (0.302 mol) of 27%
FeCl.sub.2. 250 ml of 25% NH.sub.4OH are introduced rapidly with
vigorous stirring. The mixture is stirred for 30 min. The liquors
are removed by magnetic separation. The ferrofluid is washed 3
times consecutively with 2 liters of water. The nitric ferrofluid
is stirred for 15 min with 200 ml of HNO.sub.3 [2M], and the
supernatant is removed by magnetic separation. The nitric
ferrofluid is brought to reflux with 600 ml of water and 200 ml of
Fe(NO.sub.3).sub.3 [1M] for 30 min. The supernatant is removed by
magnetic separation. The nitric ferrofluid is stirred for 15 min
with 200 ml of HNO.sub.3 [2M], the supernatant being removed by
magnetic separation. The nitric ferrofluid is washed 3 times with 3
liters of acetone, and is then taken up with 400 ml of water. The
solution is evaporated under vacuum until a final volume of 250 ml
is obtained.
TABLE-US-00002 Concentration Z ave Diameter Ms M/L nm Poly .sigma.
SQUID emu/cm.sup.3 4.85 40 nm 0.22 8.5 nm 275
EXAMPLE 2
[0142] 108 g (0.543 mol) of FeCl.sub.2.4H.sub.2O in 450 ml of
H.sub.2O is introduced into a solution of 4 liters of water and 429
ml (0.906 mol) of 27% FeCl.sub.3. 750 ml of 25% NH.sub.4OH are
introduced rapidly with stirring (1200 rpm). The mixture is stirred
for 30 min. The liquors are removed by magnetic separation. The
ferrofluid is washed twice consecutively with 3 liters of water.
The nitric ferrofluid is stirred for 1/4 h with 3 liters of
HNO.sub.3 [2M], and the supernatant is removed by magnetic
separation. The nitric ferrofluid is brought to reflux with 1300 ml
of water and 700 ml of Fe(NO.sub.3).sub.3 [1M] for 30 min (600
rpm). The supernatant is removed by magnetic separation. The nitric
ferrofluid is stirred for 15 min with 3 liters of HNO.sub.3 [2M],
the supernatant being removed by magnetic separation.
[0143] The nitric ferrofluid is washed 3 times with 3 liters of
acetone, and is then taken up with 600 ml of water. The solution is
evaporated under vacuum until a final volume of 250 ml is
obtained.
TABLE-US-00003 Concentration % yield M/L Z ave (nm) Poly .sigma.
81.8 4.45 31.3 0.21
[0144] 200 ml of the preceding solution are stirred in 2.4 liters
of HNO.sub.3 for 4 hours. The supernatant is removed by magnetic
separation. The nitric ferrofluid is washed twice with 3 liters of
acetone, and is then taken up with 400 ml of water. The solution is
evaporated under vacuum until a final volume of 250 ml is
obtained.
TABLE-US-00004 Concentration % yield M/L Z ave (nm) Poly .sigma. 77
2.742 23.3 0.20
EXAMPLE 3
##STR00010##
[0145] Step a: Diethyl-1-[diethoxyphosphoryl]vinyl phosphonate
[0146] 13 g (0.433 mol) of paraformaldehyde and 10 ml (0.097 mol)
of diethylamine are dissolved under hot conditions in 250 ml of
methanol. 24 g (8.67.times.10.sup.-2 mol) of
diethyl[diethoxyphosphoryl]methyl phosphonate are then added. The
mixture is brought to reflux for 24 hours. The reaction medium is
concentrated under vacuum. The concentrate is taken up twice with
250 ml of toluene and is then concentrated under vacuum. The oil
obtained is dissolved in 125 ml of toluene. 0.14 g of
para-toluenesulfonic acid are added. The mixture is brought to
reflux for 24 hours with a Dean-Stark trap and is then concentrated
to dryness under vacuum. The product is extracted with 500 ml of
CH.sub.2Cl.sub.2 and is then washed twice with 250 ml of water. The
organic phase is dried over MgSO.sub.4 and concentrated under
vacuum. The crude product is purified on 625 g of Merck
Geduran.RTM. silica gel (40-63 .mu.m). Elution:
CH.sub.2Cl.sub.2/acetone--50/50 (TLC--SiO.sub.2: Rf=0.45). 18.4 g
are isolated with a yield of 71%.
[0147] MS: M/z=301.4 (ES.sup.+).
Step b: Diethyl 2-[2.2-bis(diethoxyphosphoryl)ethyl] malonate
[0148] 1.6 g (0.01 mol) of diethyl malonate, 0.07 g (0.001 mol) of
sodium ethoxide and 3 g (0.01 mol) of
diethyl[diethoxyphosphoryl]vinyl phosphonate are stirred for 15 min
in 15 ml of ethanol. 5 ml of a saturated NH.sub.4Cl solution are
added to the ethanolic solution. The mixture is concentrated under
vacuum. The residue is extracted with 30 ml of ethyl acetate and
washed twice with 5 ml of water. The organic phase is dried over
MgSO.sub.4 and is then evaporated to dryness. The oil obtained is
purified on 200 g of Merck Geduran.RTM. silica. 3.8 g are isolated
with a yield of 82%.
[0149] MS: M/z=460.9 (ES.sup.+).
Step c: 4,4-Diphosphonobutanoic acid
[0150] 7 g (15.7.times.10.sup.-2 mol) of diethyl
2-[2.2-bis(diethylphosphoryl)ethyl] malonate are brought to reflux
for 8 hours in 350 ml of HCl [5N]. The brown oil obtained is
purified on 60 g of silanized silica gel 60 (0.063-0.200 mm) with
water elution. 3.6 g are isolated with a yield of 92%.
[0151] MS: M/z=249 (ES.sup.+).
EXAMPLE 4
##STR00011##
[0153] The compound (amino alcohol hydrophilic ligand) can be
prepared according to the procedure described in patent: EP 0 922
700 A1.
EXAMPLE 5
[0154] 600 mg of the compound prepared in Example 3, step c
(2.42.times.10.sup.-3 M) and 3.2 g of the compound prepared in
Example 4 (4.85.times.10.sup.-3 M) are dissolved in 20 ml of
H.sub.2O. The pH is adjusted to 6.2 with 0.1 N NaOH. 600 mg of EDCl
(3.13.times.10.sup.-3 M) and 65 mg of HOBT (4.8.times.10.sup.-4 M)
are added and the mixture is stirred at room temperature for 24
hours. The reaction medium is poured over 400 ml of IPA and stirred
for 24 hours. The precipitate is filtered, then washed in ethyl
ether and dried under vacuum.
[0155] The crude product is dissolved in the minimum of water by
adjusting the pH to 9, then is deposited over 150 ml of Amberlite
Na resin (H.sup.+ form) overnight. The product is eluted with
water. The good fractions are concentrated under vacuum. MS,
ES.sup.-: 1385.6.
EXAMPLE 6
Step a
##STR00012##
[0157] In a 500 ml three-necked flask equipped with an electrode
and a magnetic stirrer, the gem-bisphosphonate (Example 3, step c,
30 g) is dissolved in H.sub.2O (250 ml). The pH is brought to 5.7
via NaOH and the amine (11-azido-3,6,9-trioxaundecan-1-amine,
Fluka.RTM., 21.8 g) is added in a single go: the HOBT (1.72 g),
then the EDCl (21.16 g) are added. The reaction medium is stirred
for 24 hours at room temperature. The medium is evaporated until a
final volume of around 150 ml is obtained. The pH is brought to 8
via NaOH. The solution is passed over 70 ml (30 times the
theoretical amount) of Amberlite 252 Na resin (H.sup.+ -1.8 meq/ml)
in order to remove the excess amino PEG. Elution with H.sub.2O
(V.sub.recovered=300 ml).
[0158] The solution is evaporated until a final volume of around
150 ml is obtained.
[0159] The solution is passed over 140 ml (2 times the theoretical
amount) of IRA 67 resin (OH.sup.- -1.6 meq/ml) in order to remove
the excess Cl.sup.- ions. Elution with H.sub.2O
(V.sub.recovered=260 ml). The solution is evaporated until a final
volume of around 100 ml is obtained. The solution is passed over
900 g of silanized silica. Elution with 2 l of H.sub.2O, then with
2 l of an H.sub.2O/CH.sub.3OH mixture (50/50).
[0160] m=28.46 g, yield=75%, LC/MS: in ES.sup.+ at m/z=449.12.
Step b
##STR00013##
[0162] Introduced into a 1 l autoclave reactor is the azide
obtained in step a (28.26 g) previously dissolved in EtOH (350 ml).
The medium is acidified with a solution of HCl and four spatulas of
Pd/C are introduced into the solution. The reaction medium is
stirred for 4 hours at room temperature under 4 bar of hydrogen.
The reaction medium is filtered over clarcel and the solution is
evaporated to dryness until a straw yellow oil (31.21 g) is
obtained. The product is purified by passing the solution over 200
ml of IRA 67 resin (OH.sup.- -1.6 meq/ml) in order to remove the
excess Cl.sup.- ions. Elution with H.sub.2O.
[0163] m=12.25 g, yield=46% (oil), LC/MS: in ES.sup.+ at
m/z=423.12.
Step c
##STR00014##
[0165] In a 250 ml three-necked flask, equipped with a magnetic
stirrer, the intermediate prepared previously (12 g) is dissolved
in DMSO (200 ml). Triethylamine (6696 .mu.l), then diethyl squarate
(4205 .mu.l) are added. The medium is stirred for 72 hours at room
temperature. The solution is concentrated to dryness using a vane
pump until a yellow oil is obtained that is purified over silanized
silica (elution with 2000 ml of H.sub.2O, then with 2000 ml of
H.sub.2O/CH.sub.3OH (80/20), then with 2000 ml of an
H.sub.2O/CH.sub.3OH mixture (50/50) and with 1000 ml of CH.sub.3OH.
m=7.2 g, yield=46.4% (oil), LC/MS: in ES.sup.+ at m/z=547.25.
EXAMPLE 7
##STR00015##
[0167] In a pill-making machine, equipped with an electrode and a
magnetic stirrer, the intermediate prepared previously (Example 6,
step c) (0.137 g; 2.5.times.10.sup.-4 mol) is dissolved in H.sub.2O
(2 ml). The pH of the solution is adjusted to 7.5 with a saturated
Na.sub.2CO.sub.3 solution. The dye (0.05 g; 1.26.times.10.sup.-4
mol), previously dissolved in DMSO (1 ml), is added to the medium.
The pH of the solution is equal to 6.5. The pH of the solution is
brought to 8 with a saturated Na.sub.2CO.sub.3 solution. The
solution is stirred for 48 h at room temperature. The pH of the
solution is brought to 7 with a 1N hydrochloric acid solution. The
solution is evaporated to dryness using a vane pump. The oil
obtained is dissolved in H.sub.2O (10 ml) and purified by
chromatography over a cartridge of RP18 silica (25-40 .mu.m) of 90
g. 95 mg of product are isolated with a yield of 95%. LC/MS: in
ES.sup.-, m/z=860.19.
EXAMPLE 8
##STR00016##
[0169] In a pill-making machine, equipped with an electrode and a
magnetic stirrer, the compound prepared in Example 6, step c (0.137
g; 2.5.times.10.sup.-4 mol) is dissolved in H.sub.2O (2 ml). The pH
of the solution is adjusted to 7.5 with a saturated
Na.sub.2CO.sub.3 solution. The dye (0.102 g; 1.26.times.10.sup.-4
mol), previously dissolved in DMSO (1 ml), is added to the medium.
The pH of the solution is equal to 5.5. The pH of the solution is
brought to 8 with a saturated Na.sub.2CO.sub.3 solution. The
solution is stirred for 48 h at room temperature. The pH of the
solution is brought to 7 with a 1N hydrochloric acid solution. The
solution is evaporated to dryness using a vane pump. The oil
obtained is dissolved in H.sub.2O (10 ml) and purified by
chromatography over a cartridge of RP18 silica (25-40 .mu.m) of 90
g. 100 mg of product are isolated with a yield of 66%.
EXAMPLE 9
Step a
##STR00017##
[0171] 30 g of methylenebis(phosphonic dichloride) are stirred in
180 ml of toluene previously dried over molecular sieves. The
temperature is kept at 0.degree. C. A solution of 60 ml of benzyl
alcohol and of 37.5 ml of pyridine is added dropwise using a
syringe driver over 4 hours, it being necessary for the temperature
not to exceed 0.degree. C. The medium is stirred for 4 hours at RT.
The insoluble portion is removed by filtration, and rinsed several
times with toluene. The organic phase is washed 3 times with 150 ml
of 2N sodium hydroxide, 250 ml of water, dried over MgSO.sub.4,
then concentrated. The mixture is purified over silica (eluent:
heptane/ethyl acetate: 30/70).
[0172] TLC [SiO.sub.2-Hept/AcOEt: 3/7--R.sub.f=0.3]--yield:
60%>
[0173] LC/MS in ES.sup.+ 537.21
Step b
##STR00018##
[0175] The compound prepared in step a and 15C5 are stirred in 240
ml of freshly distilled THF. 1.15 g of 60% NaH are added in small
amounts to the medium. The stirring is continued for 1/2 h. t-Butyl
bromoacetate, put into 25 ml of THF, is added dropwise to the ice
bath. The mixture is stirred for 3 h at RT. The reaction medium is
concentrated under vacuum, taken up with a saturated solution of
NH.sub.4Cl and extracted with 2.times.200 ml of CH.sub.2Cl.sub.2.
The organic phase is dried over MgSO.sub.4 and purified (Si60
cartridge: 201 nm; flow rate=20 ml/min; gradient:
CH.sub.2Cl.sub.2/acetone)--yield: 65%.
[0176] LC/MS in ES.sup.+=650.65, BP-tBu: (M+1)=595.26
Step c
##STR00019##
[0178] 3.4 g of the compound from step b are put into solution in
35 ml of CH.sub.2Cl.sub.2. The solution is kept in an ice bath and
3.4 ml of TFA are added dropwise. The mixture is stirred for 4
hours at 0.degree. C., then for 20 hours at RT. The reaction medium
is evaporated under vacuum at 20.degree. C.
[0179] The product is taken up with 20 ml of CH.sub.2Cl.sub.2 and
washed with water then purified.
[0180] (RP 18 cartridge; detection at 201 nm; flow rate=20 ml/min;
gradient: water-TFA pH=2.77/CH.sub.3CN)--yield: 51%
[0181] LC/MS in ES.sup.+ 595.28
EXAMPLE 10
##STR00020##
[0183] 1 g of the compound obtained in step c from Example 3
(4.03.times.10.sup.-3 mol) and 3.26 g of PEG750
(4.43.times.10.sup.-3 mol) are dissolved in 55 ml of water. The pH
is adjusted to 6.2. 272 mg of HOBT (2.01.times.10.sup.-3 mol) are
added and the reaction medium is stirred for 5 minutes. 1.148 g of
EDCl (6.times.10.sup.-3 mol) are then added and stirring is
continued for 24 h. Purification over Amberlite 252Na resin with
fixation of the product at pH 9. 2.3 g are obtained, i.e. a yield
of 59%.
[0184] LC/MS: ES.sup.- mode, series of peaks centered about BP at
964.35.
EXAMPLE 11
Step a
##STR00021##
[0186] 150 mg of dye (1.88.times.10.sup.-4 mol) are dissolved in 15
ml of DMF. Added successively are: 60 mg of HOBT
(4.44.times.10.sup.-4 mol), 51 mg of TBTU (1.58.times.10.sup.-4
mol), 84 mg of tert-butyl protected diamine (4.15.times.10.sup.-4
mol) and 0.165 ml of DIPEA (9.4.times.10.sup.-4 mol). The reaction
medium is stirred overnight at room temperature. Purification by
reverse-phase flash chromatography. 103.4 mg of product are
isolated with a yield of 55%.
[0187] LC/MS: ES.sup.+ mode BP at 980.89
Step b
##STR00022##
[0189] 103 mg of the compound prepared in step a
(1.05.times.10.sup.-4 mol) are stirred in 3 ml of TFA/TIS/water
(proportion: 90/2.5/2.5) for 30 minutes. Purification by
reverse-phase flash chromatography. 40 mg of product are obtained,
i.e. a yield of 43%.
[0190] LC/MS: ES.sup.+ mode BP at 879.39 z=1.
Step c
##STR00023##
[0192] 26 mg of the compound obtained in step c from Example 9
(4.37.times.10.sup.-5 mol), 18 mg of DCC (8.72.times.10.sup.-5 mol)
and 8 mg of NHS (6.95.times.10.sup.-5 mol) are stirred for 3 hours
at room temperature in 5 ml of dichloromethane. The DCU is
filtered. 40 mg (4.54.times.10.sup.-5 mol) of the dye obtained in
step b and a few drops of TEA are then added. The stirring is
continued for 3 hours. Purification by reverse-phase flash
chromatography. 12 mg are isolated with a yield of 20%.
[0193] LC/MS: ES.sup.+ mode BP at 1457.33
Step d
##STR00024##
[0195] 150 mg of the compound prepared in the preceding step
(1.029.times.10.sup.-4 mol) are stirred in 4 ml of TFA/TIS/water
(proportions: 95/2.5/2.5). The stirring is continued for 3 hours at
room temperature. Purification by reverse-phase flash
chromatography. 50 mg are obtained, i.e. a yield of 45%.
[0196] LC/MS: ES.sup.- mode BP at 1094.53 (z=1)
EXAMPLE 12
Step a
##STR00025##
[0198] 200 mg of dye (2.51.times.10.sup.-4 mol) are solubilized in
20 ml of DMF. Added successively are: 80 mg of HOBT
(5.92.times.10.sup.-4 mol), 68 mg of TBTU (2.11.times.10.sup.-4
mol), 0.220 ml of DIPEA (1.255.times.10.sup.-3 mol) and 108 mg of
Fmoc-aminoethoxyethylamine (3.30.times.10.sup.-4 mol). The reaction
medium is stirred overnight at room temperature. Purification by
flash chromatography. Water/CH.sub.3CN. 178 mg of product are
isolated with a yield of 65%.
[0199] LC/MS: ES.sup.+ mode, BP at 1103.43 (z=1)
Step b
##STR00026##
[0201] 80 mg of the compound prepared in the preceding step
(9.0.times.10.sup.-5 mol) are put into solution in 6 ml of DMF
containing 20% piperidine. The stirring is continued for 1 hour at
room temperature. Purification by reverse-phase flash
chromatography. 50 mg of product are obtained, i.e. a yield of
40%.
[0202] LC/MS: ES.sup.- mode, BP at 880.09 (z=1)
Step c
##STR00027##
[0204] 20 mg of the benzyl-containing tweezers obtained in step c
from Example 9 (3.36.times.10.sup.-5 mol), 14 mg of DCC
(6.78.times.10.sup.-5 mol) and 6 mg of NHS (5.21.times.10.sup.-5
mol) are dissolved in DMF and stirred for 3 hours at room
temperature. The DCU is removed. 30 mg (3.4.times.10.sup.-5 mol) of
the dye obtained in step b and 17 .mu.l of DIPEA
(1.02.times.10.sup.-4 mol) are dissolved in 1 ml of DMF; the
activated ester is then added dropwise. The stirring is continued
for 3 hours at room temperature. Purification by reverse-phase
flash chromatography. 30 mg are obtained, i.e. a yield of 36%.
[0205] LC/MS: ES.sup.+ mode, BP at 1458.90 (z=1).
Step d
##STR00028##
[0207] 30 mg of the compound prepared in step c
(2.03.times.10.sup.-5 mol) are dissolved in 3 ml of TFA/TIS/water
(proportions: 95/2.5/2.5) for 3 h 50 min at room temperature.
Purification by flash chromatography. 6 mg are obtained, i.e. a
yield of 35%.
[0208] LC/MS: ES.sup.+ mode, BP at 1098.21 (z=1) and 550.3
(z=2).
EXAMPLE 13
Step a
##STR00029##
[0210] 50 mg of IR820 (Aldrich.RTM., 5.88.times.10.sup.-5 mol) and
14.65 mg (8.82.times.10.sup.-5 mol) of phenylboronic acid are
heated to reflux in the presence of 10.7 mg (9.24.times.10.sup.-6
mol) of tetrakis(triphenylphosphine)palladium and of 40.7 mg of
K.sub.2CO.sub.3 (2.9.times.10.sup.-4 mol) for 24 h at 110.degree.
C. At the end of the experiment, the palladium is filtered.
Purification by reverse-phase flash chromatography. 33 mg are
obtained, i.e. a yield of 62%.
[0211] LC/MS: ES.sup.+ mode, BP at 913.27 (z=1).
Step b
##STR00030##
[0213] 18 mg of the compound prepared in step a
(1.96.times.10.sup.-5 mol), 7 mg of HOBT (4.31.times.10.sup.-5
mol), 7 mg of TBTU (4.70.times.10.sup.-5 mol), 10 mg of
Fmoc-aminoethoxyethylamine (2.15.times.10.sup.-6 mol) and 11 .mu.l
of DIPEA (9.8.times.10.sup.-5 mol) are stirred overnight at room
temperature. The reaction medium is triturated in diethyl ether (20
mg) and filtered. 13 mg of product are thus isolated with a yield
of 55%.
Step c
##STR00031##
[0215] 13 mg of the compound obtained in step b
(1.06.times.10.sup.-5 mol) are stirred in DMF and 20%
piperidine.
[0216] The stirring is continued for 30 minutes at room
temperature. Precipitation into 20 ml of ethyl ether and stirring
for 1 hour at room temperature. 4.3 mg are obtained, i.e. a yield
of 40%.
Step d
##STR00032##
[0218] 20 mg of the compound obtained in step c from Example 9
(3.36.times.10.sup.-5 mol), 14 mg of DCC (6.78.times.10.sup.-5 mol)
and 6 mg of NHS (5.21.times.10.sup.-5 mol) are dissolved for 3
hours at room temperature. The DCU is removed. 35 mg
(3.4.times.10.sup.-5 mol) of the dye obtained in step c and 17
.mu.l of DIPEA (1.02.times.10.sup.-4 mol) are dissolved in 1 ml of
DMF; the activated ester is then added dropwise. The stirring is
continued for 3 hours at room temperature.
[0219] Precipitation in 50 ml of ethyl ether. 20 mg are obtained,
i.e. a yield of 37%.
Step e
##STR00033##
[0221] 20 mg of the compound prepared in step d
(1.25.times.10.sup.-5 mol) are stirred in 3 ml of TFA/TIS/water
(95/2.5/2.5) for 3 h and at room temperature. Purification by
reverse-phase flash chromatography. 4 mg are isolated, i.e. a yield
of 26%.
EXAMPLE 14
[0222] 60 .mu.mol of the compound obtained in Example 5 in solution
in 10 ml of water are added dropwise to a solution of 1 ml of
Example 2 (acid ferrofluid) at a concentration of 2.75 M/L diluted
in 100 ml of water. The mixture is stirred for 20 minutes at room
temperature and the pH is adjusted to 7.2. The solution obtained is
ultrafiltered through a membrane having a cut-off threshold of 30
kD. 300 ml of filtrate are removed in order to obtain a final
solution of 10 ml. [0223] [Fe]: 0.260 M/L PCS size=28 nm [0224]
Degree of grafting [amino alcohol compound/Fe]=2% mol/mol
EXAMPLE 15
[0225] 3 ml of the compound described in Example 2 ([Fe]=1.336
mol/l) are diluted in 100 ml of water. Added to the solution,
successively and with a delay of minutes between each addition, are
a solution of 46 mg of the compound described in Example 5 in 2 ml
of water, a solution of 3.85 mg of the compound described in
Example 7 in 2 ml of water and finally a solution of 46 mg of the
compound from Example 5 in 2 ml of water. The solution is stirred
for 15 minutes at room temperature and the pH is adjusted to 7.4
with a solution of NaOH. The medium is ultrafiltered through a 30
KD membrane and the volume of the solution is brought to 20 ml for
an iron concentration of 0.191 mol/l. PCS: 26.8.
EXAMPLE 16
[0226] According to the procedure from Example 16, various binary
or tertiary combinations of bisphosphonate compounds in variable
proportions are fixed to the particles of iron oxide described in
Example 1 or 2 as summarized in the following table:
TABLE-US-00005 Biphosphonate 1 Biphosphonate 2 Biphosphonate 3 PCS
No. Particles (% mol) (% mol) (% mol) size nm 1 Example 1 Example 5
(40) Example 3 (60) -- 42 2 Example 2 Example 5 (60) Example 3 (30)
Example 7 (10) 28 3 Example 2 Example 5 (95) Example 7 (5) -- 27 4
Example 2 Example 5 (90) Example 8 (10) -- 26 5 Example 2 Example 5
(95) Example 11 (5) -- 28 6 Example 2 Example 10 (20) Example 3
(80) -- 29 7 Example 2 Example 10 (95) Example 12 (5) -- 28 8
Example 2 Example 10 (80) Example 3 (15) Example 12 (5) 27 9
Example 2 Example 10 (98) Example 13 (2) -- 28 10 Example 2 Example
10 (100) -- -- 27 11 Example 2 Example 5 (100) -- -- 26 12 Example
2 Example 5 (96) Example 12 (2) Example 7 (2) 27 13 Example 2
Example 5 (50) Example 10 (50) -- 28
[0227] The numbers between parentheses indicate the degree of
coverage, for example for No. 1: the coverage is composed of 40% of
the compound from Example 5 (amino alcohol ligand) and of 60% of
the compound from Example 3 (bisphosphonate compound not connected
to a ligand).
* * * * *